The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition.

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins.

Introduction: Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. Methods: The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. Results: CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. Discussion: CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.

Large B-Cell Lymphomas in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms-Updated Classification and New Concepts.

The family/class of the large B-cell lymphomas (LBCL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) features only a few major changes as compared to the 4th edition. In most entities, there are only subtle changes, many of them only representing some minor modifications in diagnostic terms. Major changes have been made in the diffuse large B-cell lymphomas (DLBCL)/high-grade B-cell lymphomas (HGBL) associated with MYC and BCL2 and/or BCL6 rearrangements. This category now consists of MYC and BCL2 rearranged cases exclusively, while the MYC/BCL6 double hit lymphomas now constitute genetic subtypes of DLBCL, not otherwise specified (NOS) or of HGBL, NOS. Other major changes are the conceptual merger of lymphomas arising in immune-privileged sites and the description of LBCL arising in the setting of immune dysregulation/deficiency. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of the different entities are provided.

Localized- and advanced-stage follicular lymphomas differ in their gene expression profiles.

The genetic background of follicular lymphomas (FLs) diagnosed in advanced clinical stages III/IV, and which are frequently characterized by t(14;18), has been substantially unraveled. Molecular features, as exemplified in the clinicogenetic risk model m7FLIPI, are important tools in risk stratification. In contrast, little information is available concerning localized-stage FL (clinical stages I/II), which accounts for ∼20% of newly diagnosed FL in which the detection rate of t(14;18) is only ∼50%. To investigate the genetic background of localized-stage FL, patient cohorts with advanced-stage FL or localized-stage FL, uniformly treated within phase 3 trials of the German Low-Grade Lymphoma Study Group, were comparatively analyzed. Targeted gene expression (GE) profiling of 184 genes using nCounter technology was performed in 110 localized-stage and 556 advanced-stage FL patients. By penalized Cox regression, a prognostic GE signature could not be identified in patients with advanced-stage FL, consistent with results from global tests and univariate regression. In contrast, it was possible to define robust GE signatures discriminating localized-stage and advanced-stage FL (area under the curve, 0.98) by penalized logistic regression. Of note, 3% of samples harboring an "advanced-stage signature" in the localized-stage cohort exhibited inferior failure-free survival (hazard ratio [HR], 7.1; P = .0003). Likewise, in the advanced-stage cohort, 7% of samples with a "localized-stage signature" had prolonged failure-free survival (HR, 2.3; P = .017) and overall survival (HR, 3.4; P = .072). These data support the concept of a biological difference between localized-stage and advanced-stage FL that might contribute to the superior outcome of localized FL.

A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy.

Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. Targeting aberrantly overexpressed kinases to cure MPM is a promising therapeutic strategy. Here, we examined ALK, MET and mTOR as potential therapeutic targets and determined the combinatorial efficacy of ALK and mTOR targeting on tumor cell growth in vivo. First, ALK overexpression, rearrangement and mutation were studied in primary MPM by qRT-PCR, FISH, immunohistochemistry and sequence analysis; mTOR and MET expression by qRT-PCR and immunohistochemistry. Overexpression of full-length ALK transcripts was observed in 25 (19.5%) of 128 primary MPM, of which ten expressed ALK protein. ALK overexpression was not associated with gene rearrangement, amplification or kinase-domain mutation. mTOR protein was detected in 28.7% MPM, co-expressed with ALK or MET in 5% and 15% MPM, respectively. The ALK/MET inhibitor crizotinib enhanced the anti-tumor effect of the mTOR-inhibitor rapamycin in a patient-derived MPM xenograft with co-activated ALK/mTOR: combined therapy achieved tumor shrinkage in 4/5 tumors and growth stagnation in one tumor. Treatment effects on proliferation, apoptosis, autophagy and pathway signaling were assessed using Ki-67 immunohistochemistry, TUNEL assay, LC3B immunofluorescence, and immunoblotting. Co-treatment significantly suppressed cell proliferation and induced autophagy and caspase-independent, necrotic cell death. Rapamycin/crizotinib simultaneously inhibited mTORC1 (evidenced by S6 kinase and RPS6 dephosphorylation) and ALK signaling (ALK, AKT, STAT3 dephosphorylation), and crizotinib suppressed the adverse AKT activation induced by rapamycin. In conclusion, co-treatment with rapamycin and crizotinib is effective in suppressing MPM tumor growth and should be further explored as a therapeutic alternative in mesothelioma.

A novel, highly sensitive ALK antibody 1A4 facilitates effective screening for ALK rearrangements in lung adenocarcinomas by standard immunohistochemistry.

INTRODUCTION: Successful treatment of lung cancer patients with crizotinib depends on the accurate diagnosis of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements. The approved fluorescence in-situ hybridization test is complex and difficult to use in daily diagnostic practice. Immunohistochemical assays-rapid and perfectly adapted for routine pathology practice-have been proposed as alternatives. We evaluated the novel high affinity ALK 1A4 antibody for routine diagnostics in formalin fixed, paraffin-embedded tumor specimens. METHODS: Detection of ALK protein expression was investigated by comparing the new 1A4 antibody and the established D5F3 antibody/Ventana system in 218 lung cancer specimens with known ALK status preanalyzed by quantitative reverse transcription-polymerase chain reaction and fluorescence in-situ hybridization (20 ALK-positive cases, 198 ALK-negative cases). RESULTS: The accuracy of both immunohistochemical assays for the detection of ALK rearrangements was high. Using a conventional staining procedure without signal enhancement, the 1A4 antibody assay identified all 20 ALK-rearranged tumors (100% sensitivity) and correctly characterized 196 of 198 negative cases (99.1% specificity). The D5F3/Ventana assay detected 19 ALK-rearranged tumors and typed 217 of 218 tumors correctly (95% sensitivity, 99.5 % specificity). CONCLUSIONS: The novel 1A4 antibody represents a promising candidate for screening lung tumors for the presence of ALK rearrangements.

Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

INTRODUCTION: The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. METHODS: We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. RESULTS: qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. CONCLUSIONS: Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

Analysis of 11q22-q23 deletion target genes in B-cell chronic lymphocytic leukaemia: evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B.

Deletion of 11q22-q23 is associated with an aggressive course of B-cell chronic lymphocytic leukaemia (B-CLL). Since only in a subset of these cases biallelic inactivation of ATM was observed, we sought to identify other disease-associated genes within 11q22-q23 by analysing NPAT (cell-cycle regulation), CUL5 (ubiquitin-dependent apoptosis regulation) and PPP2R1B (component of the cell-cycle and apoptosis regulating PP2A) for point mutations and their expression in B-CLL by single-strand conformation polymorphism/sequence analysis of the transcripts and real-time polymerase chain reaction. Though none of the genes were affected by deleterious mutations, we observed a significant down-regulation of NPAT in B-CLL versus CD19+ B cells and of CUL5 in 11q deletion versus non-deletion B-CLL samples and measured reduced PPP2R1B transcript levels in a subset of B-CLL cases. Alternative splicing of PPP2R1B transcripts (skipping of exons 2/3, 3, 9) was associated with a reduced activity of protein phosphatase 2A. Together, these results implicate deregulation of the cell-cycle and apoptosis regulators NPAT, CUL5 and PPP2R1B and a role for these genes in the pathogenesis of B-CLL.

Development of a mantle cell lymphoma in an ATM heterozygous woman after occupational exposure to ionising radiation and somatic mutation of the second allele.

Predisposition to lymphomagenesis is a well-known phenomenon of ataxia-telangiectasia, a recessive disorder caused by germline inactivation of ATM. ATM encodes a protein implicated in the repair of radiation induced double-strand breaks. Biallelic ATM inactivation was described also in sporadic lymphoid malignancies, supporting a role of ATM as a tumour suppressor gene. It is, however, still unclear whether ATM heterozygotes are at higher risk of tumours. We describe an ATM heterozygous patient, who developed a mantle cell lymphoma (MCL) after occupational exposure to ionising radiation and somatic mutation of the second ATM allele supporting the contention that heterozygous germline ATM alterations in combination with irradiation exposure predisposes to sporadic MCL.

Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes.

Deletion of chromosome region 11q22-q23 defines a subgroup of patients with B-cell chronic lymphocytic leukemia (B-CLL) characterized by poor survival. Although the tumor-suppressor gene ATM in the consensus deletion region was found to be biallelically inactivated in about one third of B-CLL cases, in the majority of those who have this deletion, inactivation of the remaining ATM allele was not observed. To identify a second disease-associated gene, we investigated two B-CLL cases with translocation breakpoints in the critical 11q23 deletion region. In one case, a t(X;11)(q13;q23) was cloned and two novel genes were isolated. The breakpoint on 11q23 affected the ARHGAP20 gene, which encodes a protein predicted to be involved in the regulation of Rho family GTPases. The breakpoint on Xq13 occurred in BRWD3, which codes for a putative novel transcription factor. The rearrangement of ARHGAP20 and BRWD3 did not result in fusion transcripts, but it disrupted both genes. Mutation analysis of 28 B-CLL samples with monoallelic deletions and two B-CLL samples with 11q23 translocations detected no deleterious mutation in the remaining copy of ARHGAP20. Quantitative expression analysis in 22 B-CLLs revealed significant up-regulation of ARHGAP20 in CLL B cells, whereas BRWD3 was slightly down-regulated. Thus, deregulation of ARHGAP20 by altered gene expression or by gene disruption (but not point mutation) might be a general molecular mechanism of B-CLL leukemogenesis.